Abstract

Background: Acquired resistance to endocrine therapies is associated with disease relapse and progression. In vitro, endocrine resistance is accompanied by increased Src activity and the development of a highly invasive and migratory phenotype. Here we have investigated the role of Src as a modulator of intercellular adhesion in endocrine-resistant breast cancer cells.Methods: Src and beta-catenin expression and activity were determined in endocrine-sensitive MCF7 and T47D cells and tamoxifen-resistant ('TamR') MCF7 cell variants at their basal level and following treatment with the Src inhibitor AZD0530 (0-2µM) using Western Blotting. Cell migration was assessed by seeding cells onto matrix-coated porous membranes in the presence or absence of AZD0530. After 24hrs, migratory cells were fixed, stained and counted. The effects of Src inhibition on beta-catenin association with E-cadherin was measured by immunoprecipitation whilst changes in cell morphology were determined using differential interference contrast microscopy.Results: In contrast to endocrine-sensitive MCF7 cells, tamoxifen-resistant ('TamR') variants displayed high levels of both activated Src kinase (Src phosphorylated at Y419) and tyrosine-phosphorylated beta-catenin (Y142 and Y654). Accompanying this was a decrease in the association of beta-catenin with E-cadherin; TamR cells also grew as dispersed cell colonies and displayed a greatly enhanced migratory phenotype compared to MCF7 cells. Inhibition of Src in TamR cells significantly reduced both Src activity and levels of beta-catenin (Y142 and Y654) whilst restoring beta-catenin association with E-cadherin and suppressing cell migration. Immunohistochemistry studies involving ER+ve breast cancer tissue (n=390 from the ABC trial) have revealed that higher nuclear levels of y142 phosphorylated beta-catenin correlate with increased levels of total beta-catenin in the nuclei, suggesting src activated beta-catenin may confer an aggressive breast cancer phenotype.Treatment of ER+ve, endocrine-sensitive MCF7 and T47D cells with tamoxifen in the short term promoted an increase in Src activity and an accompanying Src-dependent increase in tyrosine-phosphorylated Y142 and Y654 beta-catenin, No effect was seen following treatment with fulvestrant or upon oestrogen deprivation.Conclusion: Src-dependent changes in beta catenin phosphorylation status appear to play a central role in the development of the migratory nature accompanying tamoxifen resistance in breast cancer cells which may correlate with findings in clinical tissue. Importantly, Src-dependent changes in beta-catenin activity appear to be promoted by tamoxifen itself during the drug-responsive phase and can be suppressed by the concomitant use of Src inhibitors.